Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.

Abbreviations used

BHQ

2,5-di-t-butyl-1,4-benzohydroquinone

cADPr

cyclic ADP-ribose

CPA

cyclopiazonic acid

ER

endoplasmic retriculum

IP3

inositol-1,4,5-trisphosphate

IP3R

inositol-1,4,5-trisphosphate receptor

PEG

polyethylene glycol

PMCA

plasma membrane Ca2+ ATPase

RyR

ryanodine receptor

SERCA

sarco-endoplasmic reticulum Ca2+ ATPase

TG

thapsigargin

TG-R

thapsigargin resistant

TG-S

thapsigargin sensitive

The endoplasmic reticulum (ER) is comprised of subcompartments with distinct functional roles in protein synthesis, protein glycosylation, Ca2+ sequestration and cell survival (Verma et al. 1990b; Takei et al. 1992; Mattson et al. 2000; Paschen and Frandsen 2001). Disrupted ER Ca2+ handling may alter protein synthesis, protein folding, and glycosylation and is implicated in both acute and chronic neurological disorders (Mattson et al. 2000; Paschen and Frandsen 2001). The ER utilizes a family of high affinity P-type calcium pumps known as sarco-endoplasmic reticulum Ca2+ ATPases (SERCA; East 2000). ER-accumulated Ca2+ can also be released into the cytoplasm in response to second messengers via specific receptor channels, such as inositol-1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR) (Berridge and Irvine 1989; Iino 1999). The expression patterns of SERCAs, IP3R and RyR display a high degree of regional heterogeneity within the brain (Verma et al. 1990b, 1992) and even within individual brain cells (Takei et al. 1992; Berridge 1998). These observations suggest that brain ER Ca2+ handling properties may be highly specialized in different brain regions.

In the present study we have utilized ATP- and Mg2+-dependent 45Ca2+ uptake in rat brain microsomes, tissue sections, and digitonin-permeabilized brain-derived cell lines to study the TG-R Ca2+ buffering compartment. We have also evaluated the contribution of this compartment to cytoplasmic Ca2+ buffering via calcium imaging studies in non-permeabilized cells. We show that the TG-R pool is primarily expressed in nervous tissue, is markedly enriched in brainstem and spinal cord, is loaded by a novel Mg2+-dependent P-type Ca2+ ATPase activity and displays unique anion permeabilities.

All reagents used in general laboratory procedures were purchased from Sigma Chemical Company (St. Louis, MO, USA). Thapsigargin was obtained from Molecular Probes (Eugene, OR, USA). [45Ca2+]CaCl2 and [γ-32P]ATP were obtained from NEN (Boston, MA, USA). The caloxin peptide (Chaudhary et al. 2001) was synthesized at the Uniformed Services University bio-instrumentation core facility. All other reagents used were of the highest grade available.

All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in a humidified incubator at 37°C in a 5% CO2 atmosphere under ATCC recommended conditions. Cerebellar granule cells were cultured from cerebella of 8-day-old Wistar rat pups as described (Nicoletti et al. 1986). For preparation of primary whole rat brain astrocytes, mixed glial cultures were initially seeded from dissected brains of male Sprague-Dawley rats and enriched to virtually pure populations of astrocytes by established procedures (Grimaldi et al. 1994). All primary astrocyte cultures were used between 1 to 2 weeks of plating (∼90% confluence).

Histochemical 45Ca2+ uptake assays were performed in serial frozen sections as described previously (Verma et al. 1990a, 1992). The uptake buffer contained 80 mm KCl and 10 mm potassium oxalate in place of potassium phosphate for better localization. Incubations were performed at 37°C for 30–60 min. Slides were transferred to ice-cold wash buffer to terminate the incubation. After washing twice for 2 min at 4°C, sections were dried under a cool air stream accompanied by vacuum aspiration of excess fluid and then exposed to beta particle-sensitive film (Kodak Biomax MR) for 24 h to image the anatomical distribution of accumulated 45Ca2+. After developing film, brain sections were wiped from the slides and the accumulated radioactivity measured in 3 mL CytoScint scintillation cocktail. For comparison of total accumulated 45Ca2+ between different tissues, sections were taken through the uptake reaction, dried, and scraped from the slides into 1-mL plastic tubes using a razor blade. Sections were then dissolved in 0.25 mL of 0.1 m NaOH and 2% sodium dodecyl sulfate. Separate aliquots were then monitored for accumulated radioactivity and for protein concentration. The results were normalized for protein.

Microsomal uptake and release of Ca2+ was measured using radiotracer 45Ca2+ in uptake assays performed in 96-well microplates as described above with modifications. Routine uptake assays were performed for 90 min in the absence and presence of TG (10 nmol/mg protein). At 90 min (which approximates uptake saturation in phosphate-supported buffer), Ca2+ ATPase inhibitors were added and the assay allowed to continue. Microsomal calcium flux was subsequently monitored by vacuum filtration at different intervals followed by scintillation counting. Non-specific uptake was considered 45Ca2+ accumulation in the presence of 10 µm A23187. Calcium leakage was defined as the decrease in specific 45Ca2+ at various times following the addition of inhibitors compared to saturation at 90 min. For optimal effects of calcium releasing agents, small volume (96-well) assays were utilized by performing 45Ca2+ uptake while including the releasing agent in the reaction from the beginning of the experiment. The difference in net calcium accumulation in the presence of these agents specifically determines the release sensitivity of microsomes to these agents (Verma et al. 1990a, 1992).

Ca2+-dependent ATPase activity was determined by measuring liberated phosphate in the filtrate using a described method (Zaidi and Michaelis 1999) with minor modifications. The assay was performed in a 20-mm HEPES-KOH (pH 7.3) buffer containing 80 mm KCl, 10 mm potassium oxalate, 5 mm sodium azide, 3% PEG and concentrations of free Ca2+ were varied by adding EGTA and using a calcium-sensitive electrode. Following fresh additions of 5 mm dithiothreitol, 1 mm MgCl2, and brain microsomes (100 µg/mL final protein), reactions were started by the addition of 2 mm ATP in a final volume of 250 µL, and continued at 37°C for 30 min. Vacuum filtration of the 96-well MAFB microplate into a polystyrene 96-well culture microplate halted the uptake assay. A solution containing 2% ammonium molybdate in 1.8 m H2SO4 and 5% (v/v) of W-1 polyoxyethylene ether was added to the filtrate (100 µL per well). A microwell plate reader was used to immediately measure the formation of colored phospho-molybdate complex in the filtrate at 410 nm. Ca2+-activated ATP hydrolysis was expressed as relative calcium-dependent increase in absorbance (Zaidi and Michaelis 1999). For determination of calcium-independent ATPase activity, chelex 100 resin (Bio-Rad) was used to treat the uptake buffer overnight and the free calcium was determined to be less than 10 nm.

U-87 cells were seeded on glass coverslips coated with poly-l-lysine whereas U-251 cells were seeded on uncoated coverslips. [Ca2+]i measurements were performed according to a previously published protocol (Grimaldi et al. 1999).

Ratio values In U-87 and U-251 were calibrated to [Ca2+]i by obtaining Fmax and Rmax and Fmin and Rmin by exposing the cells to 10 µm ionomycin in presence of 10 mm calcium. After the maximal signal was obtained, cells were perfused with calcium-free Krebs buffer containing 10 mm EGTA. [Ca2+]i was then calculated using the equation developed by Grynkiewicz et al. (1985).

Data collected was parametric in nature, and consisted of treating the same sample tissue preparations with different treatments. The primary statistical test used to analyze experimental data for significant difference was two-tailed Student's t-test for paired samples. Typically, at least three independent experiments were performed prior to data analysis. Statistical significance was considered to be p < 0.05.

To study the TG-R Ca2+ pool specifically, we utilized Mg2+- and ATP-dependent 45Ca2+ uptake in rat brain preparations in the presence of TG. The Ca2+ uptake buffer we utilized contains an ATP-regenerating system and sodium azide, an inhibitor of both cytochrome c oxidase and the F1F0 ATPase (Harris 1989), which effectively excluded mitochondria from contributing to net ATP-dependent Ca2+ sequestration (Verma et al. 1990a, 1992). Microsomes potentially contain revesicularized plasma membrane along with the plasma membrane Ca2+ ATPase (PMCA), which is not sensitive to TG inhibition. Digitonin (10 µm) maximally permeabilizes the plasma membrane without effecting Ca2+ accumulation in ER vescicles (Fiskum 1985). Digitonin (10 µm) was thus routinely included in all assays.

Microsomal 45Ca2+ uptake experiments reported in the literature are often performed with widely variable methodology. Uptake experiments are often carried out for short time periods in buffers variably containing Ca2+ precipitating anions such as phosphate or oxalate. To determine whether the presence of Ca2+ precipitating anions influenced the TG sensitivity of brain microsomes we conducted 45Ca2+ uptake assays for short periods (15 min) in buffers that contained either chloride, phosphate, or oxalate as the sole anion. Although the absolute amount of 45Ca2+ uptake increased in the presence of phosphate or oxalate, the greatest TG sensitivity was seen in oxalate buffers (Fig. 1a). About 90% of all the A23187-sensitive, oxalate-supported uptake was blocked by 10 nm TG. Inhibition of the remaining 10% required 100 µm TG. A biphasic response to TG was also seen in phosphate buffers, although the extent of inhibition was less than that seen with TG. 45Ca2+ uptake performed in chloride buffers was the least sensitive to TG inhibition.

Figure 1. TG-S and TG-R Ca2+ accumulation in rat brain microsomes. Mg2+ and ATP-dependent 45Ca2+ uptake was measured utilizing buffers in which the predominant anion was oxalate, phosphate, or chloride. (a) In uptake studies conducted for 15 min, the greatest TG sensitivity was seen in oxalate buffers (▪), followed by phosphate (•), and then chloride (◆). Note the plateau in sensitivity between 10 nm and 10 µm TG. (b) Calcium uptake was determined in the absence (▵) and presence of 100 nm TG (•). Although Ca2+ uptake is diminished in the presence of TG, a distinct TG-R component is seen. By subtracting the TG-R component from the total, a TG-S component (○) can be distinguished from the TG-R. The relative contribution of the TG-S and TG-R components to the overall Ca2+ accumulation varies markedly with the anionic composition of the buffer and the time of assay. (c) TG concentration response distinguishes two distinct components of brain microsomal Ca2+ accumulation. Phosphate-supported 45Ca2+ uptake was monitored in rat brain microsomes for 60 min in the presence of increasing concentrations of TG. A biphasic inhibition of Ca2+ uptake is again apparent, with a distinct component of Ca2+ uptake being sensitive to TG over a range from 10 pm to 10 nm. A second inhibitory action of TG on the remaining Ca2+ uptake activity is seen from 1 µm to 100 µm. The two distinct Ca2+-accumulating activities distinguished by high and low sensitivities to TG inhibition represent the TG-S and TG-R components, respectively. (d) TG-S and TG-R components are displayed separately, with their respective TG sensitivities depicted as percentage inhibition. For this representation, the TG-S component (○) was determined by subtracting Ca2+ accumulation in the presence of 10 nm TG from the total. The remaining activity represents the TG-R component (•). (e) Both the TG-S and TG-R Ca2+-accumulating activities are temperature dependent but only the TG-S component is sensitive to sulfhydryl reduction by dithiothreitol (DTT). The data represent means ± SE of three independent experiments performed in triplicate.

In experiments of longer duration, Ca2+ accumulation with oxalate as the sole cotransported anion was found to progress over a 3-h time course (Fig. 1b). By utilizing 100 nm TG (1 nmol/mg protein), a concentration that maximally inhibits SERCA activity (Lytton et al. 1991; Pizzo et al. 1997), this total Ca2+ accumulation was resolved into two distinct activities that we refer to as the TG-sensitive (TG-S) and the TG-resistant (TG-R) components (Fig. 1b). By subtracting the TG-R activity from total Ca2+ accumulation, the TG-S component could be selectively displayed. Ca2+ accumulation into the TG-S and TG-R components could be distinguished by their respective kinetic profiles. TG-S Ca2+ uptake in oxalate containing buffer appeared to saturate by 2–3 h incubation whereas TG-R Ca2+ uptake increased linearly over the 3 h of incubation. In oxalate buffers, the TG-S and TG-R components represented 86.4 and 13.6% of the total Ca2+ accumulation at 1 h, respectively. With phosphate-based buffers the TG-S and TG-R components appeared to saturate by ∼1 h. At this time point, the TG-S and TG-R components represented 60.7 and 39.3% of the total Ca2+ accumulation (Fig. 1b), with this ratio varying with time prior to saturation. Compared with oxalate, the phosphate-based buffer had lower overall Ca2+ accumulation but a greater proportion of the TG-R component. In chloride-based buffers, overall Ca2+ accumulation was further reduced; however, a minimal sensitivity to TG-inhibition was observed (Fig. 1b). Thus, detection of the TG-R component of the overall Ca2+ accumulation was highly dependent on assay times, and was more significantly revealed in buffers with phosphate and chloride as the predominant anions.

Subsequent comparisons of the TG-S and TG-R compartments were standardized and performed in phosphate-containing buffers at 60 min incubation times. Under these conditions TG again inhibited Ca2+ uptake in a biphasic manner (Fig. 1c). Over the concentration range of 10 pm−1 nm, TG produced ∼80% inhibition of the overall Ca2+ accumulation in whole brain microsomes and this represents the TG-S component described above. The potent inhibitory effect of TG is consistent with its action on all known mammalian SERCA isoforms (Lytton et al. 1991). The ∼20% of Ca2+ accumulation that remains was inhibited by TG only at micromolar concentrations. In Fig. 1(d) the amount of Ca2+ accumulation remaining in the presence of 10 nm TG was subtracted from the total to resolve the TG-S and TG-R components. Ca2+ uptake expressed as percentage inhibition of the two distinct pools clearly shows that TG-S component can be distinguished from the TG-R component by its > 1000-fold higher sensitivity to TG. Thus, the TG-S component was inhibited by TG with an IC50 of 1 nm, whereas the TG-R component had an IC50 for TG of 3 µm. Based on these results we were able to separately evaluate the biochemical properties of the TG-S and TG-R Ca2+ accumulation by performing experiments with and without the presence of 100 nm TG. Ca2+ accumulation measured in the presence of 100 nm TG was thus considered TG-R and the difference between total Ca2+ accumulation and TG-R Ca2+ accumulation was considered TG-S Ca2+ accumulation.

SERCA activity is highly sensitive to temperature and to reduction of sulfhydryl groups (Verma et al. 1990a; Scherer and Deamer 1986). Both the TG-S and TG-R components were found to be similarly sensitive to temperature (Fig. 1e), with maximum uptake occurring at 37°C. Decreasing temperature to room temperature resulted in ∼50% decrease in TG-S and TG-R Ca2+ uptake, whereas assays at 4°C virtually eliminated Ca2+ accumulation in either compartment. TG-S and TG-R Ca2+ uptake activities were, however, found to have distinct sensitivities to the sulfhydryl reducing agent dithiothreitol (Fig. 1e). Thus, removal of dithiothreitol from the uptake assay significantly diminished TG-S uptake but not TG-R uptake.

To determine whether the TG-R Ca2+ uptake in brain microsomes reflected the activity of a Mg2+-dependent ATPase, we studied the dependence of Ca2+ uptake activity on ATP and Mg2+. Figure 2 shows that Ca2+ accumulation by brain microsomes into both the TG-S and TG-R components had a similar requirement for ATP, Ca2+, and Mg2+. In all cases, the effective concentrations of these agents producing peak activity is approximately the same for both TG-S and TG-R components. Maximal uptake occurs at about 1 mm for ATP, 2 mm for Mg2+, and 5 µm for Ca2+ (Figs 2a–c). The bell-shaped dependencies of the uptake activity for the TG-S and TG-R Ca2+ uptake components has been described previously for the enzymatic activity of Ca2+ ATPases (Gould et al. 1986). The TG-S and TG-R Ca2+ uptake activities were both abolished by the calcium-specific ionophore A23187 (Fig. 2d) with an IC50 of 3 nm (data not shown). Several other ionophores, including nigericin, gramicidin and monensin, were without effect at 10 µm. Vanadate and Eosin Y, two non-specific P-type ATPase inhibitors were able to block Ca2+ uptake into both the TG-S and TG-R pools with IC50s of 100 µm and 10 µm, respectively (data not shown). The more selective ATPase inhibitors omeprazole (100 µm), ouabain (100 µm) and bafilomycin (10 µm) were without effect on either pool. To address the possible contribution of microsomal plasma membrane vesicles in the TG-R signal, we raised the digitonin concentration in our uptake assays to 100 µm, a level well above that required to permeabilize plasma membranes, but not other cell membranes (Fiskum 1985). We also utilized the newly developed selective PMCA inhibitor caloxin (Chaudhary et al. 2001; Holmes et al. 2003; De Luisi and Hofer 2003) at levels sufficient to block PMCA activity. Both approaches were without effect on TG-S or TG-R activities (Fig. 2d). To definitively rule out mitochondrial contribution towards the TG-S or TG-R signal we employed a combination of oligomycin A (100 µm) and carbonyl cyanide p-trifluromethoxy-phenylhydrazone (1 µm), which was also without effect (Fig. 2d). Together, these data suggested that the TG-S and TG-R activities were both mediated by a non-mitochondrial intracellular ATPase activity.

Figure 2. Optimal ATP, Mg2+, and Ca2+ concentrations are similar for TG-R and TG-S components. Uptake assays were performed as in Fig. 1, but over a varying range of ATP and Mg2+ concentrations (a and b, respectively). Both agents stimulated TG-S (○) and TG-R (•) Ca2+ uptake up to a maximum followed by a decline in uptake at higher concentrations. (c) Using buffers containing varying free Ca2+ levels, activation of both TG-S and TG-R Ca2+ uptake is seen up to a maximum at ∼5 µm Ca2+. Further increase in free Ca2+ produced relative inhibition for both Ca2+-accumulating activities. (d) All TG-S (white bars) and TG-R (black bars) Ca2+ uptake is abolished by the Ca2+ ionophore A23187 (10 µm) but not by 100 µm digitonin, which is known to permeabilize plasma membranes. The general P-type ATPase inhibitor, vanadate (1 mm) blocks both TG-S and TG-R uptake, whereas the PMCA inhibitor, caloxin (2 mm) and the mitochondrial Ca2+ uptake inhibitors, oligomycin (100 µm) and FCCP (1 µm) have no effect on either activity.

The SERCA inhibitors 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA) also dose-dependently inhibited brain microsome Ca2+ uptake with a suggested biphasic pattern similar to that seen with TG (Fig. 3a). BHQ and CPA were less potent than TG in inhibiting Ca2+ uptake, having IC50s of 500 nm and 5 µm, respectively, on the total Ca2+ accumulation. Similar to the effect of TG, the effect of BHQ shown in Fig. 3a leveled off between 10 and 100 µm. The plateau identified by TG concentrations between 10 nm and 1 µm appears equivalent to that displayed by BHQ. CPA also appeared to level off at the same plateau. Also, 100 µm concentrations of BHQ and CPA (1 µmol/mg protein) were not additive with TG (1 nmol/mg protein) (Fig. 3b).

Evidence for Ca2+ ATPase activity associated with the TG-R uptake was obtained by measuring ATP hydrolysis via phosphate liberation in the presence and absense of 100 nm TG. Since we sought to measure ATP activity by monitoring the production of free phosphate, an oxalate-supported buffer was utilized instead of the standard phosphate-based buffers used otherwise. ATPase activity was thus monitored via absorbance of the molybdate complex of liberated phosphate (Zaidi and Michaelis 1999). Figure 3(c) demonstrates that the total and TG-R Ca2+ ATPase components are similarly stimulated by varying concentrations of free Ca2+. Phosphate liberation from ATP seen at free Ca2+ levels below 5 nm was assumed to represent the activity of Ca2+-independent, Mg2+-dependent ATPases ubiquitous in most cellular and subcellular preparations, including microsomes. Increasing concentrations of free Ca2+ stimulated the ATP hydrolytic activity, producing a bell-shaped curve peaking at approximately 5 µm free Ca2+ for both TG-S and TG-R activities (Fig. 3c). Increasing free Ca2+ above 5 µm apparently inhibited ATPase activity, an observation similar to that reported in other brain microsomal preparations (Salvador and Mata 1998; Verma et al. 1992).

SERCAs are P-type ATPases that form a transient covalent phosphorylated enzyme intermediate as part of their Ca2+ transport cycle. Although this intermediate normally occurs at a rapid rate, the phosphoenzyme intermediate can be trapped at 4°C and precipitated with trichloroacetic acid. To explore the possible involvement of a P-type ATPase in mediating the TG-R Ca2+ accumulation, we determined whether a Ca2+-stimulated [32P]phosphoenzyme intermediate that was insensitive to SERCA inhibitors could be detected in brain microsomes. Brain microsomes were found to form TCA-precipitable [32P]phosphoenzyme intermediate in a Ca2+-stimulated manner (Figs 3d and e). In Fig. 3(d), the Ca2+-stimulated TCA-precipitable 32P-radioactivity associated with brain microsomes was counted in a beta-counter and the effects of SERCA inhibitors on this signal are shown. Counts obtained in the presence of 5 mm EGTA were negligible and have been subtracted out from all samples in Fig. 3(d). Although the SERCA inhibitors TG (500 nm), CPA (500 µm), and BHQ (750 µm) inhibit about 75% of total phosphoenzyme formation, about 25% of the phosphoenzyme activity was insensitive to these inhibitors but still sensitive to inhibition by EGTA. SDS-PAGE and autoradiography of the Ca2+-stimulated [32P]phosphoenzyme intermediate showed a diffuse band at approximately 110 kDa, consistent with the molecular weight of known SERCAs (Fig. 3e). Although EGTA abolishes 32P incorporation into this region, the SERCA inhibitors (used at the same concentrations as in Fig. 3d) are only partially effective. These results suggest that a P-type Ca2+ pump with a molecular weight similar to SERCA, but different inhibitor sensitivity, may be involved in mediating the TG-R Ca2+ accumulation.

TG treatment of cultured cells results in a rapid emptying of ER Ca2+ stores (Islam and Berggren 1993). This passive Ca2+ efflux, or leakage, from intracellular stores is poorly understood but is distinguished from Ca2+ efflux mediated by IP3R and RyR (Beecroft and Taylor 1998). To determine whether the TG-R Ca2+ sequestering compartment exhibits passive Ca2+ efflux, we performed kinetic studies in which Ca2+ accumulation was allowed to proceed for 90 min at which point 1 µm TG was added. Aliquots of the reaction were removed at different times, both during the 90-min uptake period and following TG addition. These aliquots were then filtered, washed, and counted for their 45Ca2+ content. The addition of 1 µm TG inhibits SERCA activity and resulted in a progressive efflux of calcium from the TG-S compartment (Fig. 4a). A significant portion of the accumulated calcium that does not appear to leak out of brain microsomes can be made to leak out upon the further addition of 1 mm vanadate. Similar results were seen with 1 mm eosin Y (data not shown). This presumably represents passive Ca2+ efflux from the TG-R compartment upon inhibition of the P-type ATPase that loads this compartment. Vanadate and eosin inhibit both TG-R and TG-S Ca2+ accumulation and their addition after 90 min of uptake resulted in efflux of all accumulated Ca2+ (data not shown). Initial rates of efflux appear to be similar from the TG-S and TG-R compartments. A23187 addition at any time during the experiment rapidly released (< 5 min) all sequestered Ca2+ with much faster kinetics than the spontaneous efflux. In addition to passive efflux pathways, ER Ca2+ compartments contain specific Ca2+ release channels (Berridge and Irvine 1989; Iino 1999), which allow the ligand-activated release of the sequestered Ca2+ by the second messenger IP3 (via IP3R) or by Ca2+ itself (via RyR). Although Ca2+ is believed to be the endogenous activator of RyR, high millimolar concentrations of caffeine activate release via this channel. Cyclic ADP-ribose (cADPr) and nicotinic acid-adenine dinucleotide phosphate (NAADP), have also been proposed to activate release channels located on the ER (Yamaki et al. 1998; Bak et al. 1999). To determine the sensitivity of brain TG-R and TG-S compartments to activators of IP3R and RyR, we carried out Ca2+ uptake assays in the presence and absence of TG in which were included varying doses of either IP3 or caffeine (Fig. 4b). In these assays the added release channel activator holds the channel in an open configuration during the uptake process, thus resulting in a ligand-activated leak via a specific channel (Verma et al. 1990a,b, 1992). Although both of IP3 and caffeine released calcium from the TG-S pool in a concentration-dependent manner, these agents were without effect on the TG-R pool (Fig. 4b). cADPr (10 µm) and NAADP (500 nm) released 15.3% and 6.2% of the TG-S pool calcium, respectively, but both agents were also without effect on the TG-R pool (data not shown). These results suggest that the TG-S and TG-R compartments have differential expression of Ca2+ release channels.

Figure 4. TG-S and TG-R Ca2+-accumulating compartments exhibit similar passive Ca2+ efflux pathways but have distinct sensititivities to IP3 and caffeine. Ca2+-flux studies were performed in brain microsomes as described in Methods to compare the passive efflux characteristics of the TG-S and TG-R components. At near saturation levels of Ca2+ accumulation (90 min), maximally effective TG (1 µm; 10 nmol/mg) was added to the reaction, which resulted in progressive loss of accumulated Ca2+ over time and establishment of a new steady state after an additional 30 min. The new steady state was approximately 50% of maximal saturation. Addition of vanadate (1 mm) to the reaction at 180 min to block all ATPase activity resulted in progressive release of the remaining Ca2+. Adding 10 µm A23187 at near saturation to introduce an artificial leak path for Ca2+ results in complete emptying of all sequestered Ca2+ within five min.

TG-R Ca2+ sequestration in rat brain microsomes was previously shown to be differentially expressed in distinct brain regions with higher levels seen in the brain stem and spinal cord relative to striatum and cerebral cortex (Verma et al. 1990a). We found similar differences in regional microsomes prepared from pig brain stem and spinal cord (Table 1). Microsomes from pig spinal cord had greater TG-R Ca2+ accumulation than brain stem, followed by cerebellum and forebrain. Since brain microsomes represent subcellular compartments from many different cell types, we also performed experiments in rat brain primary cell cultures of cerebellar granule cells and whole brain astrocytes. Both of these cell types demonstrate TG-R Ca2+ accumulation, with the neuronal granule cells having three times the amount demonstrated by astrocytes (Table 1). To see if human brain derived cancer cell lines also expressed TG-R Ca2+ accumulation, we determined the level of TG-R Ca2+ accumulation in rat PC-12 cells, as well as two different human neuronal and two different human glial cell lines. For these studies we employed cells permeabilized with 10 µm digitonin. In the SH-SY5Y human neuroblastoma cell line, 23.1% of the total Ca2+ accumulation is mediated by the TG-R component as compared to 29.8% in PC-12 cells. In the NBFL human neuroblastoma cell line, only 14.1% of the total Ca2+ accumulation is accounted for by the TG-R component. In the U-251 astrocytoma cell line, only 4.4% of total Ca2+ accumulation is sequestered by the TG-R component, whereas this component accounted for half of the total Ca2+ accumulation (49.1%) in U-87 astrocytoma cells. However, total amount of Ca2+ accumulation in U-251 and U-87 cell homogenates were similar.

TG-R Ca2+ uptake is variably distributed in nervous tissue-derived preparations, cell types, and cell lines. TG-S and TG-R Ca2+ uptake were analyzed as described in Methods and only the TG-R component of the total Ca2+ accumulation is reported. Mean TG-R Ca2+ uptake± SE from at least three independent experiments is shown. TG-R Ca2+ uptake in pig brain microsomes displays regional variation with higher levels in hindbrain structures. Also, TG-R Ca2+ uptake among two different cell types of the rat brain differ markedly, as shown by the three-fold higher percentage of TG-R activity in primary cerebellar granule cell cultures as compared to whole brain astrocytes. Cancer cell lines display a wide variation in the TG-R component of the overall Ca2+ uptake, ranging from almost 50% total Ca2+ uptake activity in U-87 human glioblastoma cells to a very low percentage seen in a different glioblastoma cell line, U-251 cells. Total Ca2+ uptake per mg protein, however, was similar amongst the cell lines tested.

Rat whole brain microsomes:

20.8 ± 4.8

Pig regional CNS microsomes:

Spinal cord microsomes

24.2 ± 7.1

Brainstem microsomes

17.6 ± 1.8

Cerebellum microsomes

12.1 ± 2.0

Forebrain microsomes

11.7 ± 1.2

Rat brain primary cell cultures:

Cerebellar granular cells

19.7 ± 5.6

Whole brain astrocytes

6.4 ± 2.9

Cultured cell lines:

U-87 (glioblastoma, human)

49.1 ± 6.1

PC-12 (pheochromocytoma, rat)

29.8 ± 5.4

SH-SY5Y (neuroblastoma, human)

23.1 ± 0.1

NBFL (neuroblastoma, human)

14.1 ± 1.6

U-251 (glioblastoma, human)

4.4 ± 0.8

Given the 10-fold difference in TG-R calcium pool content between the U-251 and U-87 cells, we explored the relative contribution of TG-R to cytoplasmic Ca2+ buffering in these two cell lines. Fura-2-loaded U-251 and U-87 cells were challenged with increasing concentrations of thapsigargin ranging from 2 nm to 20 µm in the absence of extracellular calcium and in the presence of 100 µm EGTA (Fig. 5a). Thapsigargin dose-dependently increased the cytosolic calcium concentration [Ca2+]i, reflecting spontaneous release from intracellular pools upon inhibition of Ca2+ pumps. In U-251 cells the maximal elevation of cytoplasmic Ca2+ by thapsigargin was seen at 20 nm, and higher concentrations did not cause any further increase of Ca2+. Conversely, in U-87 cells the effect of thapsigargin did not reach a plateau until a concentration of 2 µm, about 100-fold higher than in U-251 (Fig. 5). The inset in Fig. 5(a), which expresses the results as percentage of maximal calcium release achieved by thapsigargin in each of these two cell types, respectively, clearly demonstrates the differential sensitivity to thapsigargin seen in the two cell lines. To determine whether a significant difference in PMCA Ca2+ extrusion activity accounted for the differential responses seen in these cells, we added 2 mm caloxin to these cells while they were exposed to 20 nM TG. Caloxin addition did not result in a further elevation of [Ca2+]i. These findings demonstrate that the TG-R pool, which is not inhibited effectively by thapsigargin until concentrations of ∼1–10 µm are reached, contributes to cytoplasmic calcium buffering in intact cells.

Figure 5. Cells with high levels of TG-R require high TG concentrations to maximally release calcium from intracellular pools. (a) Fura-2-loaded U-251 (•) and U-87 cells (○) were treated with increasing concentrations of thapsigargin and monitored for changes in cytoplasmic Ca2+ levels. Thapsigargin produced an elevation of Ca2+ in U-251 cell with a maximal effect at 20 nm. A maximal effect of thapsigargin in U-87 cells was not seen until a concentration of 2 µm. U87 cells showed a higher overall level of thapsigargin induced Ca2+ elevation. The inset expresses the results as percentage maximal effect. (b) In both cell types the [Ca2+]i response seen with 20 nM TG was not altered by the PMCA inhibitor caloxin.

To determine the expression of the TG-R Ca2+-accumulating compartment in normal rat tissues, we employed 45Ca2+-uptake assays in fresh frozen sections of several different rat tissues. Fresh frozen sections accumulate 45Ca2+ in an ATP- and Mg2+-dependent manner with the uptake selectively representing sequestration by ER (Verma et al. 1990b, 1992). The sections are simply substituted for microsomes or homogenates in uptake assays and the results can be analyzed as either accumulated radioactive counts or by autoradiography to localize the signal to distinct anatomical compartments. As before, these assays contain 10 µm digitonin to permeabilize plasma membranes. For anatomical localization, best results were empirically obtained using buffers containing a combination of oxalate and chloride as the cotransported anions. The TG-R Ca2+ accumulation can reliably be measured in tissue sections of rat brain and other organs using the oxalate-chloride combined anion buffer by including TG (10 nmol/mg protein) in the uptake assays (Table 2). The highest percentage of total uptake represented by TG-R activity (20%) was seen in the brain. Similar results were also seen when 500 µm BHQ or CPA were utilized (data not shown). The adrenal gland had the next highest level of TG-R Ca2+ accumulation (18.5%) as a percentage of total Ca2+ accumulation. In cardiac muscle, 12.6% of the total uptake was TG-R, whereas in skeletal muscle, hardly any TG-R component (1.7%) could be detected. Many other organs display a TG-R component at intermediate levels ranging from 8.9% in the kidney to 0% in the urinary bladder (Table 2). These results suggest that TG-R Ca2+ accumulation may be associated with distinct organ functions and may be particularly important in nervous tissue.

Table 2. TG-I 45Ca2+ accumulation in rat brain and peripheral organs

Organ

TG-I Ca2+ accumulation (% total accumulation)

Total 45Ca2+ accumulation (% of brain)

TG-R Ca2+ uptake accounts for a much greater proportion of Ca2+ uptake activity in rat brain than in other organs. Energy-dependent Ca2+ uptake was performed using fresh frozen sections of several rat tissues in place of microsomes as described in Methods. Ca2+ uptake was monitored by scraping sections from the slides and determining accumulated radiolabel. Serial sections were subjected to treatment with A23187 (100 nmol/mg) and TG (10 nmol/mg) for the determination of the non-specific and TG-R signal, respectively. Serial sections were also processed through the uptake and wash procedures but were used for the determination of protein using concentrated protein dye as described. At least three serial sections were used for each treatment and for protein determination and less than a 3% difference was observed in values obtained from serial sections within a treatment group. Highest levels and proportions of TG-R were found in brain followed by the adrenal gland. The heart displayed intermediate levels, whereas other organs had much lower activity, with the bladder being the lowest. The relative amounts of overall calcium accumulation with respect to brain are also shown for each organ.

Brain

20.3 ± 1.6

100

Adrenal gland

18.5 ± 1.8

29.2

Heart

12.6 ± 2.5

56.4

Kidney

8.9 ± 1.0

21.4

Lung

8.1 ± 0.8

38.9

Thymus

6.8 ± 1.7

9.6

Liver

5.8 ± 1.6

16.4

Spleen

5.2 ± 0.4

39.8

Uterus

5.1 ± 3.3

6.0

Large Intestine

4.8 ± 1.4

14.6

Eye

4.6 ± 2.0

4.1

Pancreas

2.5 ± 0.3

11.3

Skin

2.1 ± 0.3

12.9

Skeletal muscle

1.7 ± 1.1

39.7

Bladder

0.0 ± 0.3

22.3

When 45Ca2+ accumulation was analyzed via autoradiography in rat brain sections, total Ca2+ accumulation showed remarkable regional heterogeneity, with distinct enrichment in specific cell layers of the cerebral cortex, hippocampus, striatum, and cerebellar cortex (Fig. 6). TG selectively inhibited Ca2+ accumulation into a number of these regions, whereas other brain regions were relatively resistant to TG inhibition at concentrations that maximally inhibit TG-S. Ca2+ accumulation in these regions thus represents the TG-R Ca2+ pool. All 45Ca2+ uptake was abolished by the Ca2+ ionophore A23187 or by omission of ATP (Figs 6c and d) so that all images obtained under these conditions were no different than the figure background. In Figs 6(e–p) the right half of each lettered section displays the TG-R component and the left half displays the total 45Ca2+ accumulation. The TG-R compartment is enriched in the deep cerebral cortex, thalamus, superior and inferior colliculi, brain stem, pontine nuclei, and deep cerebellar nuclei. Areas demonstrating very little TG-R Ca2+ accumulation include striatum, hypothalamus, substantia nigra, olfactory tubercle, and basal forebrain. Although the cerebral cortex demonstrates both TG-S and TG-R Ca2+ accumulation, the patterning of these two processes appears to be distinct. Thus, the TG-S component is selectively enriched in the superficial cortical layers, whereas TG-R is enriched in the deep cerebral cortical layers such as layer 4 and layers 6a and 6b (Figs 6e and f) In the hippocampus, TG-S and TG-R components are found in the pyramidal and dentate gyrus cell layers (Fig. 6h). In the thalamus, the ventral posterior medial nucleus (marked by an asterisk in Fig. 6h, and shown as close-up in Fig. 6i) is enriched in the TG-R pool, as is the medial geniculate nucleus (Fig. 6k). A remarkable difference in TG sensitivity is seen between the substantia nigra and other brainstem structures, such as the pontine nucleus (Figs 6j and k). This is more clearly appreciated in the close-up images of a sagittal brain section in Fig. 6(j). In the cerebellum, very high levels of TG-S are associated with the cortical layers, with highest levels in the Purkinje cell layer followed by the molecular layer (Figs 6l–n). The granule cell layer and deep cerebellar nuclei display lower levels of total Ca2+ accumulation. The highest TG-R components of cerebellar Ca2+ accumulation are associated with the deep cerebellar nuclei. Cerebellar cortical layers have lower TG-R Ca2+ accumulation. In both the rat (data not shown) and the pig spinal cord, TG sensitivity of ER Ca2+ pools was most prominent in the substantia gelatinosa and some motor nuclei (Fig. 6p), with much of the spinal cord grey matter showing TG resistance. These results reveal, for the first time, a novel compartmentation of TG-S and TG-R Ca2+ pools in the CNS. The highly unique anatomical patterns for the TG-R pool clearly differentiate it from the TG-S pool and thus provide firm evidence for two separate intracellular Ca2+-accumulating mechanisms in the brain.

Figure 6. Rat brain Ca2+ sequestering compartments with distinct sensitivities to thapsigargin are anatomically segregated. Mg2+/ATP-dependent 45Ca2+ accumulation was performed in the absence and presence of 1 µm TG using fresh-frozen rat brain sections as described in Methods. Sections were analyzed via film autoradiography to localize the 45Ca2+-accumulating compartments anatomically. Some images (i, j, o and p) were generated using film emulsion microscopic autoradiography. The coronal rat brain sections displayed proceed in a rostro-caudal direction. As shown in the saggital sections (a–d) all 45Ca2+ accumulation was abolished by 10 µm A23187 (c) or by the omission of ATP (d). TG (b) produces a marked inhibition of 45Ca2+ accumulation in many brain structures, whereas certain regions demonstrate TG-resistant 45Ca2+ accumulation. These differences are detailed in the coronal sections, which proceed in a rostral to caudal direction. The cerebral cortex of the frontal pole (FRP) in panel (e) displays much more TG-S than TG-R, whereas unique sensitivities of the main olfactory bulb (MOB) and anterior olfactory nucleus (AON) to TG are apparent. The MOB granule cell layer (MOBgr) and outer plexiform layer (MOBopl) appear to be enriched in TG-R. Within the cerebral cortex, a unique layering of the TG-R component is apparent, as seen in panels (f) and (g). Cerebral cortical layers 4, 6a, 6b are labeled. The piriform area (PIR) is enriched in TG-R whereas the anterior olfactory nucleus (AON), tenia tecta (TT), caudate-putamen (CP), olfactory tubercle (OT), and nucleus accumbens (AC) have much higher TG-S than TG-R. The septum (S) has somewhat higher proportions of TG-R than these structures. In panel (h), the hippocampus (H) and thalamus (TH) both display prominent 45Ca2+ accumulation, with significant heterogeneity seen amongst subregions. The venteropostero-medial (VPM) nucleus of the thalamus is indicated by an asterisk and a high power autoradiogram of this region is shown in panel (I). The amygdala nuclei (A) predominantly contain the TG-S pool. Panel (j) is an emulsion autoradiogram from a saggital brain section showing the marked difference in TG-sensitivity between the substantia nigra (SN) and other brainstem structures such as the pontine nucleus (P). In panel (k), the substantia nigra (SN) and ventral tegmental area (VTA) are highly sensitive to TG, whereas distinct enrichment of TG-R is seen in the medial geniculate (MG), red nucleus (RN), and specific areas of the superior colliculus (SC), such as the deep grey region (SCdg). Panels (l–n) show that although cerebellar cortex (CBC) is rich in the TG-S pool, the TG-R pool predominates in the inferior colliculus (IC), nucleus of the lateral lemniscus (NLL), rostral pontine reticular nucleus (PR), and pontine nucleus (P). In panels (m) and (n), TG-R appears to primarily count for the 45Ca2+ accumulation into the dorsal tegmental nucleus (DTN), ventral cochlear nucleus (VCO), trigeminal nucleus (V), principle sensory nucleus of the trigeminal (PSV), the olivary complex (OLV), the nucleus raphe magnus (RM), deep cerebellar nuclei (DCBN), spinal vestibular nucleus (SPV), dorsal cochlear nucleus (DCO), medial vestibular nucleus (MV), and the collection of nuclei making up the reticular formation (RN). The choroid plexus (ChP) contains both TG-S and TG-R components. White matter bundles including the corpus collosum (CC), cortical spinal tract (CS), and pyramidal tract (PY) display much lower levels of 45Ca2+ accumulation. Panel (o) shows microscopic emulsion autoradiography of the cerebellar cortex. Panel (p) shows ER 45Ca2+ accumulation in sections of pig spinal cord. The most prominent TG-sensitive structure in the cord is the substantia gelatinosa (SG).

We have described a novel intracellular CNS Ca2+ sequestering compartment that is resistant to thapsigargin, a compound routinely employed as a potent inhibitor of all known SERCA isoforms. Ca2+ uptake buffer anion composition and assay times are important in identifying the TG-R Ca2+ pool in microsomal preparations (Fig. 1). This elucidation may clarify many discrepancies in previous studies performed with varying methodologies. Although microsomal Ca2+ accumulation requires the presence of cotransported anions (Kemmeret al. 1987), very little is known about the nature of ER-associated anion transporters. Possible ER entry pathways for chloride (Martin et al. 1998; Qian et al. 1999), phosphate (Ng et al. 1993; Meissner and Allen 1981; Meng et al. 2000) and oxalate (Meng et al. 2000), which may facilitate Ca2+ uptake, have been suggested. Our findings suggest that unique anion permeabilities are associated with TG-S and TG-R compartments and we therefore predict a heterogeneous distribution of ER anion transporters in the brain and spinal cord. The temperature dependency (Fig. 1e) of TG-R Ca2+ accumulation and its absolute requirement for ATP, Mg2+, and Ca2+ (Fig. 2) support the involvement of a Ca2+ ATPase. Similar optimal concentrations of ATP, Mg2+, and Ca2+ for both TG-S and TG-R uptake suggest that both TG-S and TG-R uptake mechanisms would be mutually active under similar intracellular conditions. The differential redox sensitivity of the TG-S and TG-R compartments (Fig. 1e) may however favor uptake into the TG-R pool under oxidizing conditions. Since an oxidizing environment is necessary for disulfide bond formation during protein folding in the ER, the TG-R may represent an ER subcompartment specialized to simultaneously support protein folding concomittantly with Ca2+ transport. However, microsomal system may also contain lysosomes and/or secretory vesicles and it is possible that one of these compartments may account for the TG-resistant calcium pool. Given the widely differing methodologies used in studying calcium pools, the assay conditions we have defined here should prove useful in future explorations of the TG-R pool.

The insensitivity of TG-R Ca2+ transport to inhibition by TG, BHQ and CPA (Fig. 3), argues against the involvement of known mammalian SERCA isoforms. Both the TG-S and TG-R activities are, however, inhibited by vanadate, suggesting the involvement of a P-type ATPase in calcium sequestration into both compartments. This is further supported by our demonstration of TG-resistant Ca2+ ATPase and Ca2+-activated phosphoenzyme intermediate activities in brain microsomal preparations (Figs 3c–e). Several mammalian ion-motive P-type ATPases, in addition to SERCAs, are known to transport specific cations. These include the PMCA, Na+/K+ ATPase, and the H+/K+ ATPase. The lack of sensitivity of the TG-R pool to digitonin, caloxin, and several other specific ATPase inhibitors, as well as Na+, K+, and H+ ionophores rules out the involvement of the PMCA or other non-Ca2+ specific ion-motive mechanisms. No specific inhibitors are known for the recently identified Fe2+ (Baranano et al. 2000) and Cu2+-transporting mammalian P-type ATPases (Camakaris et al. 1999), or the mammalian homologue of the yeast Mn2+-transporting P-type ATPase (Hu et al. 2000; Mitchell et al. 2001). A novel SERCA isoform that is insensitive to TG inhibition and displays a unique intracellular localization was also recently discovered in paramecium (Kissmehl et al. 1998), although no mammalian homologues of this pump have yet been identified. It is possible that one of these mechanisms may be responsible for the TG-R Ca2+ accumulation. It is also possible that TG insensitivity results not from the presence of a unique ATPase but from a post-translational modification of a known SERCA isoform. In either case, a putatively novel or uniquely modified ATPase would still confer unique properties to calcium pools and would contribute to intracellular Ca2+ homeostasis, since in all of our assays the free Ca2+ concentration was carefully titrated to reflect normal baseline cytoplasmic free Ca2+ levels.

The observed slow release of accumulated calcium from microsome following inhibition of uptake by TG or vanadate (Fig. 4a) suggests that a common leak mechanism for Ca2+ exists on both the TG-S and TG-R compartments. The observation that 1 µm TG only released a portion of the total accumulated calcium has important implications for all studies that utilize TG in studying intracellular calcium regulation. TG treatment of cells and tissues is expected to induce passive efflux from the TG-S compartment, as well as Ca2+ influx from the extracellular environment, in an attempt to refill the depleted Ca2+ stores (Putney and Ribeiro 2000). The TG-R pool may contribute significantly to the overall cytoplasmic Ca2+ response in such experiments. This notion is supported by very high TG concentration required to elevate cytoplasmic Ca2+ in the U-87 cells (Fig. 5), which have a very high TG-R content (Table 1). However, despite the lack of effect of the PMCA inhibitor caloxin, it is possible that the differential [Ca2+]i responses of these two cell lines to TG arise in part from differences in Ca2+ extrusion mechanisms. We also found the brain microsomal TG-R Ca2+ pool to be resistant to activators of IP3R or RyR (Fig. 4b). Heterogeneous distribution of the TG-R pool could thus have contributed to the differential cytoplasmic calcium responses to TG and IP3 mobilizing agonists reported in different cell lines (Razani-Boroujerdi et al. 1994). However, the reported sensitivity of salivary gland TG-R pools to IP3 and cADPr (Ghosh et al. 1991; Beecroft and Taylor 1998) suggests that Ca2+ release from this compartment may be differentially regulated in different organs. It is also possible that specific Ca2+ release agents acting on the TG-R pool are yet to be fully appreciated (Hardy et al. 1995).

Although the use of microsomal fractions, sodium azide, digitonin, low nanomolar free [Ca2+] in the assay buffer, and freeze–thawed tissue preparations rules out plasma membrane and mitochondrial contribution to TG-R Ca2+ accumulation, further work is needed to determine whether the TG-R pool represents a subset of ER (Waldron et al. 1995, 1997) or the activity of other microsomal compartments, such as secretory vesicles (Mitchell et al. 2001) or the Golgi apparatus (Pinton et al. 1998). In either case, it is likely that the TG-R pool makes a major contribution to the overall intracellular Ca2+ buffering in some brain regions. This mechanism should be particularly important in discrete CNS regions, such as the specific layer of the cerebral cortex, certain nuclei of the thalamus, as well as brainstem and spinal cord structures (Table 2, Fig. 6). The discrete segregation of the TG-S and TG-R pools in the brain provides strong support for distinct biological roles for these two pools. The unique physiological properties of specific brain regions may in fact rely upon the unique biochemical properties of the TG-R Ca2+ pool.

The opinions and assertions contained herein are the private ones of the author, and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. Supported by NIH grant NS37814 and Department of Defense grant MDA905-02-2-0001 (to AV).